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. 2003 Jan;111(1):109-19.
doi: 10.1172/JCI15719.

src homology 2 domain-containing tyrosine phosphatase SHP-1 controls the development of allergic airway inflammation

Tohru Kamata  1 , Masakatsu Yamashita, Motoko Kimura, Kaoru Murata, Masamichi Inami, Chiori Shimizu, Kaoru Sugaya, Chrong-Reen Wang, Masaru Taniguchi, Toshinori Nakayama
Affiliations

src homology 2 domain-containing tyrosine phosphatase SHP-1 controls the development of allergic airway inflammation

Tohru Kamata et al. J Clin Invest. 2003 Jan.

Abstract

Th2 cells are generated from naive CD4 T cells upon T cell receptor (TCR) recognition of antigen and IL-4 stimulation and play crucial roles in humoral immunity against infectious microorganisms and the pathogenesis of allergic and autoimmune diseases. A tyrosine phosphatase, SHP-1, that contains src homology 2 (SH2) domains is recognized as a negative regulator for various intracellular signaling molecules, including those downstream of the TCR and the IL-4 receptor. Here we assessed the role of SHP-1 in Th1/Th2 cell differentiation and in the development of Th2-dependent allergic airway inflammation by using a natural SHP-1 mutant, the motheaten mouse. CD4 T cells appear to develop normally in the heterozygous motheaten (me/+) thymus even though they express decreased amounts of SHP-1 (about one-third the level of wild-type thymus). The me/+ naive splenic CD4 T cells showed enhanced activation by IL-4 receptor-mediated signaling but only marginal enhancement of TCR-mediated signaling. Interestingly, the generation of Th2 cells was increased and specific cytokine production of mast cells was enhanced in me/+ mice. In an OVA-induced allergic airway inflammation model, eosinophilic inflammation, mucus hyperproduction, and airway hyperresponsiveness were enhanced in me/+ mice. Thus, SHP-1 may have a role as a negative regulator in the development of allergic responses, such as allergic asthma.

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Figures

Figure 1
Figure 1
Phenotypically normal naive CD4 T cells developed in me/+ mice. (a) Representative CD4/CD8 profiles of splenocytes and thymocytes of wild-type (+/+) and heterozygous (me/+) mice with the motheaten mutation. The percentages of cells in each quadrant are shown. The numbers of cells harvested (yield) were 1.41 × 108 splenocytes in wild-type, 1.52 × 108 splenocytes in me/+, 1.72 × 108 thymocytes in wild-type, and 1.70 × 108 thymocytes in me/+. (b) Splenic CD4 T cells from wild-type and me/+ mice were cultured with IL-4 (100 U/ml) for 24 hours, and the expression of common-γ and IL-4Rα chain was assessed. Background staining is shown as hatched areas. (c) SHP-1 protein expression in splenic CD4 T cells from me/+ and wild-type mice. Immunoprecipitation and immunoblotting with anti–SHP-1 mAb were performed. Ratios of the intensities of the SHP-1 bands are shown.
Figure 2
Figure 2
TCR-induced MAPKK activation and IL-4–induced STAT6 phosphorylation in splenic CD4 T cells from me/+ mice. (a) Phosphorylation status of Erk1 and Erk2 in splenic CD4 T cells was assessed 5–60 minutes after co-cross-linking of TCR and CD4 molecules. The cells were lysed, and the lysates were subjected to immunoblotting with anti–phospho-Erk antibody. Densitometric measurements of the phosphorylated bands (p44 for Erk1 and p42 for Erk2) are shown under each band in arbitrary units. Erk1-P, phospho Erk-1. (b) IL-4–induced phosphorylation of STAT6. Splenic CD4 T cells from wild-type and me/+ mice were stimulated with IL-4 (100 U/ml) at 37°C for the indicated times. The cell lysates were subjected to immunoprecipitation with anti-STAT6 antiserum, and to immunoblotting with anti-phosphotyrosine mAb (upper panel) or anti-STAT6 antiserum (lower panel). Densitometric measurements of the phosphorylated STAT6 bands and STAT6 protein bands are shown under each band in arbitrary units. STAT6–P, phospho-STAT6.
Figure 3
Figure 3
Increased Th2 cell differentiation in me/+ naive CD4 T cells. Naive CD4 T cells from wild-type and me/+ mice were stimulated for 5 days with immobilized anti–TCR-β mAb (1 μg/ml) in the presence of 20 U/ml exogenous IL-2 and 3 U/ml IL-4 (Th2-skewed condition) or 1 U/ml IL-12 and anti–IL-4 mAb (Th1-skewed condition). The generation of Th1 and Th2 cells was assessed by cytoplasmic staining with IFN-γ and IL-4. The absolute numbers of cells harvested were similar in these cultures. The percentages of cells in each quadrant are shown. Four independent experiments were performed with similar results.
Figure 4
Figure 4
Increased Th2 cytokine production in me/+ T cells. Splenic CD4 T cells were stimulated with immobilized anti–TCR-β for the indicated times, and the concentrations of cytokines (a, IL-4; b, IL-13; and c, IL-5) in the culture supernatant were determined by ELISA. Mean values and standard deviations are shown. In several cases, the deviations are too small to be visible. Three independent experiments were performed, and similar results were obtained.
Figure 5
Figure 5
Increased IL-6 and IL-13 production of BMMCs from me/+ mice. (a) c-kit expression and IgE binding capacity of BMMCs prepared from wild-type and me/+ mice. Shaded areas represent background staining. (b) Cross-linking stimulation of FcεRI was performed by anti-DNP IgE and DNP-BSA. The production of IL-6 and IL-13 in the culture supernatant was assessed by ELISA. Mean values + SD are shown. P values of IL-6 data, from left (0 ng/ml stimulation) to right (10 ng/ml stimulation), are 0.001, 0.004, 0.027, and 0.068, and those of IL-13 data are 0.0004, 0.0006, 0.0012, and 0.0047.
Figure 6
Figure 6
OVA-induced eosinophilic infiltration in BAL fluid and airway inflammation in me/+ mice. The absolute cell number (a) and percentage (b) of eosinophils (Eosino.), lymphocytes (Lympho.), neutrophils (Neutro.), and macrophages (Mφ) in BAL fluid are shown with SDs. Seven wild-type mice and eight me/+ mice were used in this experiment. The results were obtained using the values of cell counting, percentage of the cells, total cell number per milliliter, and volume of BAL fluid recovered. *P < 0.0005, **P < 0.0001, and other P values > 0.04 by Student t test. (c–h) Eight-week-old wild-type mice (c–e) and me/+ mice (f–h) were used. (d, e, g, and h) OVA immunization and inhalation with OVA aerosol were done as in a. (c and f) Control treatment with PBS was also done. The lungs were fixed and stained with hematoxylin and eosin. ×100, c, d, f, and g; ×200, e and h. Sections shown are representative of ten lung sections per mouse from five mice in each group. Peribronchiolar mononuclear cell infiltrates are noted in the wild-type control groups (open arrowheads in d and e). The infiltrate was extended in me/+ mice (arrows in g and h). Numbers of infiltrated mononuclear cells in the perivascular and peribronchiolar regions were 159.3 ± 17.0 in the me/+ and 77.3 ± 10.7 in the wild-type group. (i–k) Lungs from me/+ mice immunized with alum alone and without OVA inhalation (i), wild-type mice treated with OVA-alum immunization and OVA inhalation (j), and me/+ mice treated with OVA-alum immunization and OVA inhalation (k) were stained with PAS. A representative staining pattern in each group is shown. ×100. i.p., intraperitoneally.
Figure 7
Figure 7
OVA-induced airway hyperresponsiveness in me/+ mice. (a) Eight-week-old me/+ mice sensitized with OVA were subjected to OVA inhalation on days 14 and 16. One day after the last OVA inhalation, airway hyperresponsiveness in response to increasing doses of methacholine was measured in a whole-body plethysmograph. Mean values of the percent above base line are shown with SDs for four mice. A total of four independent experiments were performed with similar results. P > 0.5 for methacholine stimulation (1 mg/ml and 3 mg/ml), and P < 0.05 for methacholine stimulation (10 mg/ml and 30 mg/ml), by Student t test. (b) Eight-week-old me/+ mice were treated intraperitoneally with PBS, alum, or OVA-alum. Half of the mice with OVA-alum immunization were subjected to OVA aerosol on days 14 and 16. On day 17, methacholine-induced airway hyperresponsiveness was measured. Mean values of the percent above base line are shown for a mouse in each group. Two sets of experiments were done with similar results. penh, enhanced pause. inh., inhalation.
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